Orange Fluorescent Proteins: Structural Studies of LSSmOrange, PSmOrange and PSmOrange2 Sergei Pletnev 1,2 *, Daria M. Shcherbakova 3 , Oksana M. Subach 3,6 , Nadya V. Pletneva 5 , Vladimir N. Malashkevich 4 , Steven C. Almo 4 , Zbigniew Dauter 2 , Vladislav V. Verkhusha 3 * 1 Leidos Biomedical Research Inc., Basic Research Program, Argonne, Illinois, United States of America, 2 Macromolecular Crystallography Laboratory, National Cancer Institute, Argonne, Illinois, United States of America, 3 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 4 Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America, 5 Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation, 6 Department of Nano, Bio, Information and Cognitive Technologies, Moscow Institute of Physics and Technology, Moscow, Russian Federation Abstract A structural analysis of the recently developed orange fluorescent proteins with novel phenotypes, LSSmOrange (l ex /l em at 437/572 nm), PSmOrange (l ex /l em at 548/565 nm and for photoconverted form at 636/662 nm) and PSmOrange2 (l ex /l em at 546/561 nm and for photoconverted form at 619/651 nm), is presented. The obtained crystallographic structures provide an understanding of how the ensemble of a few key mutations enabled special properties of the orange FPs. While only a single Ile161Asp mutation, enabling excited state proton transfer, is critical for LSSmOrange, other substitutions provide refinement of its special properties and an exceptional 120 nm large Stokes shift. Similarly, a single Gln64Leu mutation was sufficient to cause structural changes resulting in photoswitchability of PSmOrange, and only one additional substitution (Phe65Ile), yielding PSmOrange2, was enough to greatly decrease the energy of photoconversion and increase its efficiency of photoswitching. Fluorescence of photoconverted PSmOrange and PSmOrange2 demonstrated an unexpected bathochromic shift relative to the fluorescence of classic red FPs, such as DsRed, eqFP578 and zFP574. The structural changes associated with this fluorescence shift are of considerable value for the design of advanced far-red FPs. For this reason the chromophore transformations accompanying photoconversion of the orange FPs are discussed. Citation: Pletnev S, Shcherbakova DM, Subach OM, Pletneva NV, Malashkevich VN, et al. (2014) Orange Fluorescent Proteins: Structural Studies of LSSmOrange, PSmOrange and PSmOrange2. PLoS ONE 9(6): e99136. doi:10.1371/journal.pone.0099136 Editor: Maria Sola, Molecular Biology Institute of Barcelona, CSIC, Spain Received March 13, 2014; Accepted May 8, 2014; Published June 24, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The coordinates and structure factors for LSSmOrange, PSmOrange and PSmOrange2 have been deposited to Protein Data Bank under the accession codes 4Q7R, 4Q7T and 4Q7U. Funding: Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. W-31-109-Eng-38. The authors thank Rafael Toro for help with the crystallization screening and X29 beamline staff at NSLS for help with data collection. This work was supported in part with Federal funds from the National Cancer Institute, NIH, under contract HHSN261200800001E, the Intramural Research Program of the NIH, the NIH grants GM073913 and CA164468 (to VVV), the Albert Einstein Cancer Center grant CA013330, the New York Structural Genomics Research Center (to SCA), and by a grant from the Russian Science Foundation 14-14-00281. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products or organizations imply endorsement by the US Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] (SP); [email protected] (VVV) Introduction Fluorescent proteins (FPs) of the GFP-like family have become valuable tools for molecular biology, biochemistry, and biomed- icine. Most challenging task of the FPs studies was the development of FPs with longer excitation/emission wavelength. This pursuit was motivated by advantages of red-shifted FPs, namely, lower background of cellular autofluorescence in micros- copy, lower light scattering, and reduced tissue absorbance of longer wavelengths for in vivo imaging. In addition to common FPs, there are proteins of other phenotypes available, including FPs with a large Stokes shift (LSS) and irreversibly and reversibly photoswitchable (PS) FPs [1]. According to their emission wavelength, red-shifted FPs could be divided in the following groups: 520–540 nm yellow FPs (YFPs), 540–570 nm orange FPs (OFPs), 570–620 nm red FPs (RFPs), and .620 nm far-red FPs. Red shift of fluorescence of these FPs is predominantly achieved by extension of the conjugated system of the chromophore and its protonation/ deprotonation. The variety of spectral properties of FPs, such as excitation and emission wavelength, quantum yield, brightness, photoswitchability, Stokes shift of fluorescence, result from different chromophore structures and its interactions with surrounding amino acid residues. OFPs fill up a spectral gap between YFPs and RFPs and enable four-color imaging together with blue, green, and far-red FPs. The spectral properties of OFPs mainly result from their chromophore structures. The chromophores of OFPs are formed by conservative -X-Y-G- tri-peptides in which X = Thr, Lys, Ser or Cys. In most cases, their orange emission is attributed to a three-ring chromophore structure with the third cycle formed by cyclization of the side chain of the X residue, extending the p-electron system of the chromophore [2]. Recombinant OFPs, reported so far, have been engineered from four wild-type proteins: zFP538 (Zoanthus PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e99136
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Orange Fluorescent Proteins: Structural Studies ofLSSmOrange, PSmOrange and PSmOrange2Sergei Pletnev1,2*, Daria M. Shcherbakova3, Oksana M. Subach3,6, Nadya V. Pletneva5,
Vladimir N. Malashkevich4, Steven C. Almo4, Zbigniew Dauter2, Vladislav V. Verkhusha3*
1 Leidos Biomedical Research Inc., Basic Research Program, Argonne, Illinois, United States of America, 2 Macromolecular Crystallography Laboratory, National Cancer
Institute, Argonne, Illinois, United States of America, 3 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States
of America, 4 Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America, 5 Shemyakin–Ovchinnikov Institute of
Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation, 6 Department of Nano, Bio, Information and Cognitive Technologies, Moscow Institute
of Physics and Technology, Moscow, Russian Federation
Abstract
A structural analysis of the recently developed orange fluorescent proteins with novel phenotypes, LSSmOrange (lex/lem at437/572 nm), PSmOrange (lex/lem at 548/565 nm and for photoconverted form at 636/662 nm) and PSmOrange2 (lex/lem
at 546/561 nm and for photoconverted form at 619/651 nm), is presented. The obtained crystallographic structures providean understanding of how the ensemble of a few key mutations enabled special properties of the orange FPs. While only asingle Ile161Asp mutation, enabling excited state proton transfer, is critical for LSSmOrange, other substitutions providerefinement of its special properties and an exceptional 120 nm large Stokes shift. Similarly, a single Gln64Leu mutation wassufficient to cause structural changes resulting in photoswitchability of PSmOrange, and only one additional substitution(Phe65Ile), yielding PSmOrange2, was enough to greatly decrease the energy of photoconversion and increase its efficiencyof photoswitching. Fluorescence of photoconverted PSmOrange and PSmOrange2 demonstrated an unexpectedbathochromic shift relative to the fluorescence of classic red FPs, such as DsRed, eqFP578 and zFP574. The structuralchanges associated with this fluorescence shift are of considerable value for the design of advanced far-red FPs. For thisreason the chromophore transformations accompanying photoconversion of the orange FPs are discussed.
Citation: Pletnev S, Shcherbakova DM, Subach OM, Pletneva NV, Malashkevich VN, et al. (2014) Orange Fluorescent Proteins: Structural Studies of LSSmOrange,PSmOrange and PSmOrange2. PLoS ONE 9(6): e99136. doi:10.1371/journal.pone.0099136
Editor: Maria Sola, Molecular Biology Institute of Barcelona, CSIC, Spain
Received March 13, 2014; Accepted May 8, 2014; Published June 24, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone forany lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The coordinates and structure factors forLSSmOrange, PSmOrange and PSmOrange2 have been deposited to Protein Data Bank under the accession codes 4Q7R, 4Q7T and 4Q7U.
Funding: Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under ContractNo. W-31-109-Eng-38. The authors thank Rafael Toro for help with the crystallization screening and X29 beamline staff at NSLS for help with data collection. Thiswork was supported in part with Federal funds from the National Cancer Institute, NIH, under contract HHSN261200800001E, the Intramural Research Program ofthe NIH, the NIH grants GM073913 and CA164468 (to VVV), the Albert Einstein Cancer Center grant CA013330, the New York Structural Genomics Research Center(to SCA), and by a grant from the Russian Science Foundation 14-14-00281. The content of this publication does not necessarily reflect the views or policies of theDepartment of Health and Human Services, nor does the mention of trade names, commercial products or organizations imply endorsement by the USGovernment. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
sp.) [3], KO (Fungia concinna) [4], DsRed (Discosoma sp.) [5] and
phiYFP (Phialidium) [6] (Figure 1, Table 1).
zFP538 is an obligate tetramer of low brightness with -K-Y-G-
chromophore triad. Cyclization of Lys is accompanied by a
cleavage of the polypeptide chain between the chromophore and
the preceding amino acid residue, F65 [7–9]. Multiple rounds of
directed evolution of zFP538 yielded its bright monomeric version,
mPapaya1 [10], useful both as a protein tag and as a donor in
orange-red Forster resonance energy transfer (FRET) pairs. The
chromophore of phiYFP consists of -T-Y-G- residues. Its crystal
structure shows that the side chain of Thr does not form a third
cycle present in zFP538 and its yellow-orange emission was
attributed to the antiparallel stacking between Y203 and the
chromophore [11]. Two monomeric variants of phiYFP with
improved folding, preserving this structural feature, phiYFPv and
TagYFP, have been reported and described in [11]. The
chromophore of KO is formed by -C-Y-G- amino acids and
cyclization of Cys results in the third cycle, extending the
chromophore conjugation system [12]. However, unlike in
zFP538, the cyclization does not result in the cleavage of the
polypeptide backbone. The monomeric form of KO with an
improved folding, mKO, has been reported and characterized [4].
mKO has been further used to generate two variants with the
improved brightness, mKO2 [13] and mKOk [14]. Most prolific
FP, which yielded a total of eight OFPs, is DsRed [5]. Red
emission of its -Q-Y-G- chromophore was attributed to the
presence of acylimine group connecting the imidazolinone ring of
the chromophore with the preceding amino acid residue (F65)
extending its conjugation system [5]. Variation of the DsRed
chromophore tripeptide composition resulted in three OFPs:
mHoneydew (-M-W-G-), mBanana (-C-Y-G-) and mOrange (-T-
Y-G-) [2]. mOrange was shown to be the brightest and was used
for generation of its more photostable variant mOrange2 [15].
Additionally, DsRed was used to obtain an RFP with low
cytotoxicity, DsRed-Express2 [16,17], that served as a template
for generation of its low cytotoxic orange variant E2-Orange [18].
Recently, three OFPs with novel phenotypes have been
developed: permanently fluorescent LSSmOrange with a large
Stokes shift ( lmaxex 437 nm, lmax
em 572 nm) [19] and two proteins
photoswitchable from orange to far-red fluorescent states PSmOr-
ange ( lmaxex 548 nm, lmax
em 565 nm; photoconverted form lmaxex
636 nm, lmaxem 662 nm) [20] and PSmOrange2 ( lmax
ex 546 nm,
lmaxem 561 nm; photoconverted form lmax
ex 619 nm, lmaxem 651 nm)
[21].
Figure 1. Evolution of the subfamily of orange fluorescent proteins. (A) Phylogenic tree showing the history of the development of differentorange fluorescent proteins. (B) Chemical structures of the chromophores found in orange fluorescent proteins.doi:10.1371/journal.pone.0099136.g001
X-Ray Structures of LSSmOrange, PSmOrange and PSmOrange2
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LSSmOrange fills a spectral gap between green-yellow and red
LSSFPs. Its brightness is five-fold larger than that of the brightest
red LSSFP and is similar to that of green-yellow LSSFPs.
LSSmOrange was shown to be useful for multicolor imaging,
simultaneous detection of two Forster resonance energy transfer
(FRET)-based biosensors using a single excitation wavelength, and
EGFP - brightness relative to EGFP (product of WF and Emol compared to the brightness of EGFP (53,000 M21 cm2160.60) [52]).doi:10.1371/journal.pone.0099136.t001
X-Ray Structures of LSSmOrange, PSmOrange and PSmOrange2
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crystallization conditions was carried out using a Mosquito
Robotic Crystallization System (TTP LabTech Ltd). Large-scale
crystallization was set up by the hanging drop vapor diffusion
method at room temperature. The best crystals for LSSmOrange
were obtained from Wizard I crystallization kit (Emerald
BioSystems), condition 48 (0.2 M Zn acetate, 0.1 M acetate buffer
pH 4.5, 20% PEG 1000). Suitable for data acquisition crystals of
PSmOrange and PSmOrange2 were obtained using the ComPAS
crystallization suite (Qiagen), conditions C4 (55% MPD) and B8
(0.1 M HEPES, pH 7.5, 20% PEG 10000), respectively, in vapor
diffusion sitting drop configuration.
Data AcquisitionFor LSSmOrange, crystals X-ray diffraction data were collected
at the Advanced Photon Source on SER-CAT 22-ID beamline
(Argonne National Laboratory). Diffraction intensities were
registered on a MAR 300 CCD detector (Rayonix). Prior to data
collection the crystals were incubated in a cryoprotecting solution
consisting of 20% glycerol and 80% of well solution for 30 seconds
and were flash-frozen in 100 K nitrogen stream. Cryogenic
temperature was maintained by a CryoJetXL cooling device
(Oxford Cryosystems). Diffraction images were indexed, integrated
and scaled with the HKL2000 software [22].
Diffraction data of the PSmOrange and PSmOrange2 crystals
were collected on a Quantum 315 CCD detector (Area Detector
Systems) on the X29A beamline (National Synchrotron Light
Source, Brookhaven National Laboratory). PSmOrange and
PSmOrange2 crystals were mounted directly from the screening
trays. Prior to freezing, 20% glycerol was added to PSmOrange2
crystals as cryoprotectant. Intensities were integrated using
HKL2000 and reduced to amplitudes using TRUNCATE
[23,24]. Data processing statistics are given in Table 2.
Structure Solution and RefinementThe structure of LSSmOrange was solved by molecular
replacement method with MOLREP [25] using as a search model
a single monomer of mOrange (PDB ID: 2H5O, [8]), excluding its
chromophore. Structure refinement was performed with RE-
FMAC [26,27] and COOT [28]. Manual structure rebuilding and
addition of ordered solvent molecules were done using COOT.
Structure validation was performed with COOT and PRO-
CHECK [29].
Table 2. Data collection and refinement statistics.
Protein PSmOrange PSmOrange2 LSSmOrange
Space group P1 P21 P21
Unit cell parameters
a, b, c (A) 45.4, 50.7, 52.4 45.7, 43.9, 52.5 37.4, 107.4, 56.6
a, b, c (u) 94.7, 90.0, 106.6 90.0, 94.4, 90.0 90.0, 102.2, 90.0
Resolution range (A) 30.0–1.95 30.0–1.30 30.0–1.40
R-workc 0.2 0.148 0.147
R-freec 0.266 0.177 0.175
R.m.s.d. bond lengths (A) 0.018 0.014 0.014
R.m.s.d. angles (u) 2.15 1.59 1.98
R.m.s.d. chirality (A3) 0.13 0.08 0.159
R.m.s.d. planarity (A) 0.011 0.007 0.01
Ramachandran statistics (%)
(for non-Gly/Pro residues)
most favorable 92.7 93.9 95
additional allowed 7.3 6.1 5
aData in parentheses are given for the outermost resolution shells: 1.98–1.95 A for PSmOrange, 1.32–1.30 A for PSmOrange2, and 1.45–1.40 A for LSSmOrange.bRmerge = ShklSj |Ij(hkl) – ,I(hkl).|/ShklSj|,I(hkl).|, where Ij is the intensity measurement for reflection j and ,I. is the mean intensity over j reflections.cRwork/(Rfree) = S ||Fo(hkl)| – |Fc(hkl)||/S |Fo(hkl)|, where Fo and Fc are observed and calculated structure factors, respectively. No s-cutoff was applied. 5% of thereflections were excluded from refinement and used to calculate Rfree.doi:10.1371/journal.pone.0099136.t002
X-Ray Structures of LSSmOrange, PSmOrange and PSmOrange2
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The structures of PSmOrange and PSmOrange2 were deter-
mined by molecular replacement using PHASER [30]. Model
building and refinement were performed with REFMAC and
COOT. The quality of the final structure was verified with
composite omit maps, and the stereochemistry was checked with
MOLPROBITY [31]. The refinement statistics for LSSmOrange,
PSmOrange and PSmOrange2 are given in Table 2.
Results
LSSmOrange and PSmOrange have been engineered from
mOrange and PSmOrange2 has been engineered from PSmOr-
ange using directed molecular evolution. The resulted OFPs
constitute the following mutants: mOrange/R17H/A44V/F83L/
W143M/I161D/M163L/G196D (LSSmOrange), mOrange/
S21T/Q64L/F99Y/L124M/K162R/P186S (PSmOrange), and
PSmOrange/R17H/R36H/F65I/Q188L/A217S/G219A
(PSmOrange2) (Figures 2, 3). Here and below the numbering is
adopted from mOrange PDB file, 2H5O [8]. Note that not all of
the listed mutations are located in the nearest chromophore
environment. In fact, only three residues in LSSmOrange (M143,
D161, and L163), one in PSmOrange (L64), and two in
PSmOrange2 (I65 and S217) are situated close enough to the
chromophore to directly affect its electronic properties (Figure 3).
Crystal Structures of LSSmOrange, PSmOrange, andPSmOrange2
The asymmetric units of LSSmOrange and PSmOrange crystals
contain two monomers oriented approximately parallel to each
other, whereas the asymmetric unit of PSmOrange2 contains a
single monomer. Crystallographic symmetry operations do not
complete monomers to classic GFP-like tetramers indicating a true
monomeric nature of the proteins. Multiple conformations are
observed for the total of 30 (6.5%) amino acid residues of
LSSmOrange and for 26 (11.3%) amino acid residues of
PSmOrange2. Multiple conformations for PSmOrange were not
modeled due to the relatively low (1.94 A) resolution of the
diffraction data.
The final models of LSSmOrange, PSmOrange, and PSmOr-
ange2 have low deviations of bond length, angles, chiral volumes
and planes from ideal values indicating its high quality. In all
models, over 90% of the residues are located in the most favorable
regions of the Ramachandran plot with the rest located in the
additionally allowed regions (Table 2). Superposition of parental
mOrange with LSSmOrange, PSmOrange and PSmOrange2 by
Ca atoms resulted in root mean square deviations of 0.35 A,
0.39 A, and 0.38 A, respectively, indicating close similarity of the
structures.
Chromophores of LSSmOrange, PSmOrange, andPSmOrange2 and their Environment
Electron density maps show that LSSmOrange, PSmOrange,
and PSmOrange2 have the same chromophore structures as
parental mOrange [8]. It consists of p-hydroxyphenyl, imidazoli-
none, and 2-hydroxy-dihydrooxazole rings. The latter one results
from cyclization of T66 side chain. The chromophore adopts cis-
conformations and in all examined FPs has a noticeable but not
severe deviation from coplanarity between the adjacent p-
Figure 2. Amino acid alignment of mOrange, LSSmOrange, PSmOrange and PSmOrange2. The chromophore-forming tri-peptides arehighlighted in yellow.doi:10.1371/journal.pone.0099136.g002
X-Ray Structures of LSSmOrange, PSmOrange and PSmOrange2
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X-Ray Structures of LSSmOrange, PSmOrange and PSmOrange2
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Figure 3. Amino acid differences between the parental and successor proteins in 3D. (A) The transformation of mOrange intoLSSmOrange. (B) The transformation of mOrange into PSmOrange. (C) The transformation of PSmOrange into PSmOrange2.doi:10.1371/journal.pone.0099136.g003
Figure 4. The differences in the immediate chromophore environment between the parental and successor proteins. (A) Thedifference between mOrange and LSSmOrange. (B) The difference between mOrange and PSmOrange. (C) The difference between PSmOrange andPSmOrange2.doi:10.1371/journal.pone.0099136.g004
X-Ray Structures of LSSmOrange, PSmOrange and PSmOrange2
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hydroxyphenyl and imidazolinone rings (x1, the rotation around
Ca2-Cb2 bond, is ,5–10u). The largest deviation from coplanar-
ity is observed between 2-hydroxy-dihydrooxazole and imidazo-
linone rings of the chromophore. The Ca1 atom of the
chromophore has partially pyramidal geometry with the sum of
the valent angles around it of ,348u. Due to a distorted geometry
of Ca1 atom, these rings are oriented at the angle of ,30u with
respect to each other. The deviation of Ca1 atom from planar sp2-
geometry is presumably caused by a local steric strain between the
chromophore and F65 (preceding amino acid residue).
Key substitutions in LSSmOrange responsible for its ,135 nm
large Stokes shift comprise I161D, M163L, and W143M enabling
the excited state proton transfer (ESPT) [19]. These substitutions
introduce noticeable changes in the nearest chromophore envi-
ronment of LSSmOrange (Figure 4A). X-ray structure shows that
W143M replacement provides a shift of the side chain of L165
towards protein interior, and both W143M and M163L substitu-
tions improve the chromophore planarity relative to parental
mOrange (Figure 4A). The x1 angles of mOrange and LSSmOr-
ange chromophores are 12.3u and 6.3u, respectively. In both
mOrange and LSSmOrange, the oxygen atom of p-hydroxyphenyl
group of the chromophore forms an H-bond with the side chain
hydroxyl of S146. Rotation of the chromophore around Ca2-Cb2
bond in LSSmOrange, arising from W143M and M163L
mutations, causes the change of S146 side chain conformation.
The hydroxyl atom of S146 now forms two H-bonds, one with the
chromophore and another with the side chain of D161 (I161 in
mOrange). As a result, S146 acts as a mediator for ESPT from p-
hydroxyphenyl ring of the chromophore to carboxyl group of
D161 (Figure 4A). Four other substitutions, R17H, A44V, F83L,
and G196D, are located far from the chromophore and
presumably improve folding efficiency, brightness and photosta-
bility (Figure 3A).
The key substitutions enabling PSmOrange photoconversion
are Q64L and F99Y introduced in a process of molecular
evolution via random and rational mutagenesis of parental
mOrange [20]. In mOrange, Q64 forms H-bonds with guanidine
group of R95 and the main chain carbonyl of I60 (Figure 4B).
Substitution of Q64 with Leu disables formation of these two H-
bonds (Figure 4B). The side chain carboxyl of Y99 forms new
water-mediated H-bond with the main chain carbonyl of I60.
These two changes result in the protein that in the presence of
oxidizing agents such as potassium ferricyanide or intracellular
oxidants and light could be converted into a far-red emitting form
[20]. Out of six substitutions made in mOrange to obtain
PSmOrange Q64L is the only one located close enough to the
chromophore to cause structural changes enabling photoconver-
sion. Five other substitutions, S21T, F99Y, L124M, K162R, and
P186S, lie far away from the chromophore, presumably affecting
its folding and brightness (Figure 3B).
Analogously to PSmOrange, in PSmOrange2, position 64 is
occupied by Leu pointing out at the importance of a hydrophobic
residue in this position. PSmOrange2 differs from PSmOrange by
total six substitutions, only two of which, F65I and A217S, are
located close to the chromophore (Figure 3C). Replacement of
aromatic F65 (present in both mOrange and PSmOrange) by
aliphatic I65 is accompanied by a dramatic decrease of the
amount and intensity of light required for an efficient photo-
conversion. Four other residues are located on the surface of the
protein. S217 in PSmOrange2, forms a strong H-bond with
catalytic E215 and both, S217 and E215, adopt two conformations
(Figure 4C). In conformation ‘‘A’’, the side chain of S217 is
oriented away from E215 and forms a H-bond with the main
chain carbonyl of Y72. This makes E215 move away from the
chromophore, causes break of a H-bond between Oe2 atom of
E215 and N2 atom of the chromophore (4.4 A), and provides
formation of a new H-bond between the side chains of E215 and
Q42. In conformation ‘‘B’’, S217 points towards E215, forming a
strong H-bond (2.6 A) with its side chain and pushing E215 closer
to the chromophore, facilitating H-bonding between E215 and
nitrogen of imidazolinone ring (3.1 A) that connects it with the
protein matrix. In parental PSmOrange, position 217 is occupied
by Ala, as a result, E215 is moved away from the chromophore
forming no H-bond with it (E215-Oe2 – TYG-N2 distance is
3.9 A) (Figure 4C).
Photoconverted far-red forms of PSmOrange and PSmOrange2
are relatively unstable and undergo 50% degradation within
several days after photoconversion, presumably due to the overall
oxidation and degradation of the protein [20,21]. This instability
made impossible obtaining the suitable crystals of the far-red
forms. Photoconversion of the crystals of PSmOrange and
PSmOrange2 attempted in crystallization drops with added
potassium ferricyanide was also unsuccessful presumably due to
a poor diffusion of the oxidant in the crystals.
In an earlier work on PSmOrange, it was shown that mass-
spectrometry analysis of the photoconverted chromophore-con-
taining peptide was in a good agreement with the mechanism of
PSmOrange photoswitching involving a cleavage of the polypep-
tide chain between the main chain carbonyl and Ca of F65 [20]. It
has been suggested that the PSmOrange2 photoconversion is
similar and may consist of a break of the polypeptide chain
between the main chain carbonyl and Ca of I65. [21]. To get an
idea of the structural changes taking place in far-red forms of
PSmOrange and PSmOrange2, we have modeled their structures
based on the corresponding non-photoconverted proteins
(Figure 5). The modeling revealed that the cleavage of Ca-C
Figure 5. The structures of the PSmOrange and DsRed chromophores. (A) The structure of the orange form of the PSmOrangechromophore. (B) The modeled structure of the photoconverted far-red form of the PSmOrange chromophore. (C) The structure of the DsRedchromophore.doi:10.1371/journal.pone.0099136.g005
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bond of F65/I65 eliminates the strain imposed on the chromo-
phore by internal a-helix of the protein, causing Ca1 atom of the
chromophore to adopt a true planar sp2-geometry (Figure 5B). As
a result, the oxazole ring of the chromophore moves away from
F65/I65 and becomes positioned in–plane with two other rings,
significantly improving the efficiency of the overall chromophore
conjugation. Oxidation of the hydroxyl of 2-hydroxy-dihydroox-
azole to carbonyl puts this group in plane with the rest of the
chromophore; it faces (but is not embedded in) the hydrophobic
pocket formed by the residues F14, V16, A44, L46, F/I65, and
Y120. Appearance of C = O group extends the chromophore
conjugation system, providing an additional contribution to the
red shift of the fluorescence of the photoconverted PSmOrange
and PSmOrange2.
Discussion
Continuing progress in fluorescence imaging requires probes
with additional colors and properties optimized for emerging
imaging techniques. A typical process for the development of FPs
with desired properties includes rational design followed by
random mutagenesis. Rational design relies on the accumulated
knowledge about the influence of the certain amino acid residues
in the immediate chromophore environment on FP properties. At
this stage, first key mutations responsible for the desired target
properties of the FP are introduced. This minimally essential
variant is then subjected to a directed molecular evolution aimed
at optimization of an overall performance of FP.
Development of monomeric variant of KO started from the
introduction of seven point mutations on the surface of the protein
(F102S, A104S, V123T, C151S, F162Y, F193Y, and G195S)
known to disrupt the tetrameric interfaces of DsRed [32]. Here
and below the numbering for amino acids is adopted from original
publications. Additional mutagenesis that followed found two
other sets of mutations, twelve (K11R, V25I, K32R, S55A, T62V,
Q96E, E117Y, V133I, S139V, T150A, A166E, and Q190G) and
three (F13Y, C115T, and C217S), to improve mKO’s brightness
and folding efficiency, respectively [4] (Figure 6).
The development of monomeric FPs of different colors
collectively named mFruits was also started from the disruption
of the interfaces of tetrameric DsRed to yield mRFP1.1 [2] that
was further used as a template for the development of monomeric
FPs of multiple colors [2]. Figure 6 shows the key residues
providing orange emission of FPs derived from DsRed. Y67W is
the key substitution converting red mRFP1.1 into orange FP,
mHoneydew. Two substitutions, M66C and Q213L, are required
to transform mRFP1.1 in mTangerine, which in turn can be
converted into mBanana by additional I197E mutation. Four
point mutations, V7I, M182K, M66T, and T195V, are required
to convert mRFP1.1 in the OFP prototype, mOFP.T.8. Two
additional mutations, T41F and L83F, converted mOFP.T.8 in a
highly acclaimed bright OFP mOrange. In all cases, rational
design has been followed by directed evolution approach to perfect
newly generated FPs - enhance their brightness, folding efficiency,
and photostability.
The other vivid example of rational design and directed
evolution approach combination is mPapaya1 [10] (Figure 6).
First, to design monomeric version of zFP538, four mutations
(I106R, V115E, D164K, and R178H) were introduced at the
interfaces between its subunits (mPapaya0.2). Then, to restore the
brightness, lost during monomerization, the protein was subjected
to four rounds of directed evolution that introduced seven
additional mutations (mPapaya0.27). To further improve mono-
meric character of mPapaya0.27, additional three mutations,
Y127R, C149T, and S166K were made resulting in truly
monomeric mPapaya0.6. Finally, to improve photostability
mPapaya0.6 was subjected to additional cycle of directed evolution
resulting in mPapaya0.6/F99Y/Y168C designated as mPapaya1.
Bright monomeric FPs serve as templates for the development
of FPs with special properties, such as FPs with a large Stokes shift,
photoswitching FPs and Fluorescent timers [1]. Currently
available LSSFPs include green T-Sapphire [33], yellow mAme-
trine [34], orange LSSmOrange [19], and red LSSmKate1 [35],
Figure 6. An evolution of orange fluorescent proteins derived from KO, DsRed and zFP538 with mutations critical to the phenotypeof each variant.doi:10.1371/journal.pone.0099136.g006
X-Ray Structures of LSSmOrange, PSmOrange and PSmOrange2
PLOS ONE | www.plosone.org 9 June 2014 | Volume 9 | Issue 6 | e99136
LSSmKate2 [35], and mKeima1 [36]. Engineering of LSSFPs
aims at providing an ESPT between Tyr hydroxyl of the
chromophore and its nearest environment. The common mech-
anism implies that the chromophore is initially protonated. Upon
excitation, the proton is transferred to a nearby proton acceptor
either directly or with the help of an H-bond network. The anionic
chromophore intermediate emits a photon, returns to the ground
state, and becomes protonated.
In wild-type GFP, two ESPT pathways have been reported: the
major one follows a well-defined chromophore-Water22-S205-
E222 proton wire [37], while a less efficient one is based on a
change of T203 conformation promoting proton ejection out of
the b-barrel [38]. In the ground state, the chromophore is
reprotonated by E222 acquiring a proton from the outside by the
E5 entry pathway, located near the N-terminus of GFP [38]. In
green T-Sapphire and yellow mAmetrine, ESPT follows the same
pathway as the major one of wtGFP. In LSSmKate1, ESPT is
realized as the proton shuttle between the cis-chromophore and
E160, whereas in LSSmKate2, ESPT pathway comprises trans-
chromophore, S158, and D160 [39]. Similarly, in mKeima,
proton is transferred along the proton wire cis-chromophore-S146-
D161 [40], [41]. All elements of the mKeima proton wire are
present in LSSmOrange (cis-chromophore) indicating to a similar
mechanism of ESPT; this suggestion has been confirmed by site-
directed mutagenesis. Earlier, we have demonstrated that both
LSSmOrange/S146A and LSSmOrange/D161A mutants disturb
LSS phenotype and LSSmOrange/D161A displays a regular
Stokes shift ( lmaxex / lmax
em 550/565 nm) similar to that of mOrange
[19]. Parental mOrange has position 146 already occupied by Ser,
which is one of two essential residues required for ESPT.
PSFPs are widely used for tracking intracellular proteins,
organelles, and individual cells [42], [43]. Fluorescence of PSFPs
is switched from one color to another by the light of a specific
wavelength. A majority of PSFPs including Dendra2 [44], mEos2
[45], Kaede [46], mKikGR [47], mClavGR2 [48] and their
derivatives change their fluorescence from green to red after
irradiation with a relatively phototoxic violet light (3902410 nm).
The blue-to-green proteins photoswitchable with this light, PSCFP
and PSCFP2, are also available [44]. Photoconversion of
PSmOrange and PSmOrange2 with 480–540 nm light makes
these proteins the first PSFPs efficiently photoswitched with non-
phototoxic visible light [20], [21]. SDS-PAGE and mass
spectrometry results demonstrated that PSmOrange photoconver-
sion is accompanied by a cleavage of polypeptide chain between
the chromophore and F65 and by oxidation of the hydroxyl of 2-
hydroxy-dihydrooxazole ring of the chromophore to carbonyl
[20]. This modification strongly affects the conjugation system of
the chromophore - it enables the chromophore to recover its
distorted planarity around Ca1 atom and adds C = O bond to the
conjugation resulting in a substantial (,90 nm) bathochromic shift
of photoconverted PSmOrange absorbance/emission bands
(Figure 5B). This is not the case for the other RFPs, such as
DsRed (Discosoma sp.) ( lmaxex / lmax
em 558/583 nm) [5], eqFP578
(Entacmaea quadricolor) ( lmaxex / lmax
em 552/578 nm) [49], or zFP574
(Zoanthus sp.) ( lmaxex / lmax
em 553/574 nm) [50]: in all these RFPs,
carbonyl of the amino acid preceding the chromophore is not
coplanar with it (Figure 5C). In fact, for these proteins a ,80udihedral angle between the carbonyl group and the chromophore
makes the conjugation extremely inefficient. This carbonyl cannot
be coplanar with the chromophore due to the bent in the central
a-helix embedding the chromophore. Inefficient conjugation
results in a blue-shift of fluorescence of DsRed, eqFP578, and
zFP574 relative to that of photoswitched PSmOrange.
It has been demonstrated that position 64 mutation is minimally
required for appearance of photoswitchable properties [20]. In
mOrange, Q64 forms an H-bond with R95; this H-bond is absent
in PSmOrange, where Q64 is replaced with hydrophobic Leu
(Figure 4B), suggesting that the absence of H-bond between
residues 64 and 95 facilitates orange to far-red photoconversion of
PSmOrange chromophore.
Monomeric PSmOrange2 has been developed as an improved
version of PSmOrange with faster and more efficient photoswitch-
ing. Its photoconversion could be achieved with common two-
photon lasers and it is, therefore, much more user-friendly than
parental PSmOrange. Moreover, PSmOrange2 could be used as
an acceptor in Forster resonance energy transfer with green
fluorescent donors. This fact together with its high efficiency of
photoconversion and fast photoswitching kinetics enabled photo-
switching of PSmOrange2 via FRET from the donor FP. The
brightness of orange and far-red forms of PSmOrange2 is 1.9-fold
and 1.2-fold lower than that of respective PSmOrange forms
(Table 1). However, its photoswitching contrast, which is 9-fold
higher than that of PSmOrange, a substantially higher efficiency of
PSmOrange2 photoconversion than that of the parental protein
makes PSmOrange2 a better tag. The key mutation that provided
dramatic improvement of PSmOrange2 photoconversion is F65I.
Replacement of aromatic F65 with aliphatic I65 and decrease of
the light energy required for photoswitching imply that I65
facilitates backbone cleavage and decreases the activation barrier
of photoconversion. Modeling of photoconverted PSmOrange
structure revealed that the residue 65 and the oxidized chromo-
phore move away from each other after the cleavage of Ca1-C
bond (Figure 5B). A lower energy required for a photoconversion
of PSmOrange2 suggests that less bulky side chain of I65 enables
an easier stabilization of detached Ile within its immediate
environment than of a bulkier and more rigid F65 in PSmOrange.
Substitution A217S increases quantum yield of the orange form of
PSmOrange2 [21]. This is presumably caused by an increased
charge separation in the chromophore, taking place as a result of a
new H-bond between E215 and its N2 atom. Formation of this H-
bond occurs only in the presence of S217 that makes E215 move
closer to the chromophore. A similar H-bond between N2 and
E215 has been described earlier for rsTagRFP where it enabled
formation of a fluorescent zwitter-ionic species [51].
Conclusions
Here we presented the structural analysis of three recently
developed OFPs with novel phenotypes such as a large Stokes shift
and photoswitchability. Spectroscopic characteristics provided the
insights in the properties of these OFPs and extensive mutagenesis
identified the residues responsible for the photochemical changes.
Crystallographic structures presented here revealed how the
ensemble of key mutations introduced in the parental FPs enabled
those changes.
For LSSmOrange, ESPT occurs over the cis-chromophore-
S146-D161 pathway and is caused by three key substitutions:
I161D, M163L, and W143M. The W143M replacement provided
a shift of the side chain of L165 towards protein interior, and both
W143M and M163L improve the chromophore planarity.
Rotation of the chromophore around x1 angle, arising from these
two mutations, causes the change of S146 side chain conforma-
tion. As a result, S146 acts as a mediator for ESPT from p-
hydroxyphenyl ring of the chromophore to carboxyl group of
D161.
For PSmOrange and PSmOrange2, Q64L mutation is mini-
mally required for appearance of photoswitchable properties. The
X-Ray Structures of LSSmOrange, PSmOrange and PSmOrange2
PLOS ONE | www.plosone.org 10 June 2014 | Volume 9 | Issue 6 | e99136
structural data demonstrated that this mutation disrupts an H-
bond between positions 64 and R95 facilitating orange to far-red
photoconversion of PSmOrange chromophore. The photoconver-
sion itself is accompanied by cleavage of Ca1-C bond of F/I65,
resulting in recovery of the distorted chromophore planarity, and
by oxidation of OH-group of 2-hydroxy-dihydrooxazole to C = O,
resulting in the extension of the chromophore conjugation system.
This was the primary source of a substantial (,90 nm) bath-
ochromic red shift of fluorescence in photoconverted PSmOrange
and PSmOrange2. Substitution A217S in PSmOrange2 induces
the shift of E215 closer to the chromophore and cause formation
of its H-bond with N2 atom, increasing polarization of the
chromophore and its quantum yield.
The detailed analysis of the chromophore structures and
chromophore transformations in LSSmOrange and PSmOr-
ange/PSmOrange2 will serve as a basis for engineering of future
advanced FPs (Figure 7). First, enhanced OFP variants could be
designed by introducing ESPT or photoconversion pathways in
OFPs of different origins including wild-type OFPs. The key
positions for mutations (Figure S1) can be used to introduce
substitutions in new protein templates at the first step, followed by
random mutagenesis. Second, novel OFP phenotypes can be
obtained by combining different pathways of OFP chromophore
transformations in a single FP. We anticipate that the LSS
phenotype can be combined with PS phenotypes to engineer PS
LSS OFP. This future FP should further advance the multicolor
photolabelling in wide-field fluorescence microscopy and in super-
resolution imaging of live cells. Lastly, another engineering goal
could be a development of a FP with the autocatalytic formation of
the photoconverted far-red PSmOrange chromophore. Such
permanently fluorescent far-red FP is highly desirable as a
genetically-encoded probe for non-invasive deep-tissue in vivo
imaging.
The coordinates and structure factors were deposited in the
Protein Data Bank under the accession codes 4Q7R, 4Q7T and
4Q7U for LSSmOrange, PSmOrange and PSmOrange2, respec-
tively.
Supporting Information
Figure S1 Amino acid alignment of orange fluorescentproteins. The chromophore-forming tri-peptides are highlighted
in yellow.
(DOC)
Author Contributions
Performed the experiments: SP DMS OMS NVP VNM SCA ZD VVV.
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